50 ESTIMATION OF CHROMATIN ABNORMALITY OF OGYE ROOSTER SEMEN WITH Diff-Quik STAINING

2016 ◽  
Vol 28 (2) ◽  
pp. 155
Author(s):  
S. W. Kim ◽  
A. R. Choi ◽  
C. Y. Choe ◽  
D. K. Kim ◽  
H. H. Seong ◽  
...  
Keyword(s):  
Staining Method ◽  
Sperm Quality ◽  
Sperm Head ◽  
Head Movements ◽  
Sperm Chromatin ◽  
Frozen Semen ◽  
Sperm Nuclei ◽  
Live Sperm ◽  
The Relationship ◽  
Fresh Semen

The abnormality of Ogye rooster sperm chromatin could be detected by simple sperm staining. In this abstract, a Diff-Quick staining kit was tested for assessment of chicken sperm quality. Using a standard bright-field microscope, Diff-Quik stains can be reproducibly, easily, and routinely monitored with simple staining. The presence of abnormal chromatin staining of rooster sperm was determined by darker stain in head. In the fresh semen, the viabilities of 3 tested Ogye spermatozoa were 93.53, 82.42, and 90.63%, and normal chromatin rates were 87.96, 74.25, and 85.10%, respectively. However, after cryopreservation, the rates of viability of thawed semen were reduced to 69.58, 61.98, and 72.20%, and normal chromatin rate also reduced to 58.91, 48.49, and 63.34%. A significant correlation between live sperm and normal sperm nuclei was 0.875 in fresh semen and 0.513 in frozen semen. After incubation of sperm at 37°C for 5 min, the rates of viability, chromatin normality, and sperm head activity were shown as 90.63 ± 1.28%, 82.44 ± 8.09%, and 66.68 ± 10.29% in fresh semen. However, the rates of thawed semen were reduced to 67.92 ± 7.55%, 56.92 ± 12.15%, and 47.32 ± 5.02%, respectively. The relationship between chromatin normality and sperm head movements in fresh and thawed semen were 0.564 and 0.540, respectively. With these results, the chicken sperm normality could be assessed by the Diff-Quik staining, which could be used for chromatin status of sperm head and activated morphology of live spermatozoa, as a simple and rapid staining method.

scholarly journals Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

Reproduction ◽  
2012 ◽  
Vol 143 (6) ◽  
pp. 799-813 ◽  
Author(s):  
G A Montano ◽  
D C Kraemer ◽  
C C Love ◽  
T R Robeck ◽  
J K O'Brien
Keyword(s):  
Bottlenose Dolphin ◽  
Sperm Quality ◽  
Critical Evaluation ◽  
Membrane Integrity ◽  
Ex Situ ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Freezing Method ◽  
Frozen Semen ◽  
Motility Parameters

Artificial insemination (AI) with sex-sorted frozen–thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen–thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen–thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze–thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

226 DAY LENGTH DOES NOT AFFECT LIVE SPERM NUCLEAR SHAPE IN THE STALLION

2008 ◽  
Vol 20 (1) ◽  
pp. 192
Author(s):  
J. J. Parrish ◽  
C. Mueller ◽  
E. Ludwig ◽  
J. L. Susko-Parrish
Keyword(s):  
Sperm Head ◽  
Nuclear Shape ◽  
Day Length ◽  
Fourier Harmonic ◽  
Glass Slides ◽  
Large Numbers ◽  
Alternative Approach ◽  
Fertility Data ◽  
Sperm Nuclei ◽  
Live Sperm

Fourier harmonic analysis (FHA) of sperm nuclei is a precise and objective method to evaluate shape of the sperm head, with the calculated harmonic amplitudes highly related to male fertility. The FHA approach has been developed for use in the bull and the boar but has not yet been applied to the stallion. Direct utilization of the previous fluorescent approaches to identify and image live sperm nuclei in the bull cannot be used in the stallion due to the increased thickness of the post-nuclear region and thin anterior region of the sperm head. An alternative approach was developed in which live and motile sperm were isolated after filtration of an ejaculate through a Sephadex G-15 column. The resulting live sperm were sonicated briefly to separate tails and heads. The heads were isolated on a 45–90 discontinous Percoll gradient, fixed with paraformaldehyde (0.2%), centrifuged onto glass slides, and dried. The slides were then stained with eosin (1%), cleared with water, and dried again; Permount was added, followed by a coverslip. Slides were imaged with phase contrast microscopy; digital images were acquired and evaluated with custom software to identify perimeter coordinates of sperm nuclei. The perimeter coordinates were next converted to Fourier harmonic amplitudes 0–5 (HA0–HA5) using trigonomic regression at 1 degree equally spaced angles. Fertility of bulls were previously reported to be most related to changes in HA0 and HA2 but no information is available on stallions. As fertility data on stallions is limited, to evaluate FHA in the equine, the day length (period of light) was increased in January from the ambient 9–10 h to 16. Semen samples from 5 light horse stallions were collected at weekly intervals for 8 weeks following the increase in light. It was hypothesized that fertility would increase for each stallion over the course of the experiment, as testosterone increases and spermatogenesis improves with increasing day length, as previously shown. Each week semen samples were evaluated for FHA live sperm nuclei, as described above. All parameter means and variations were recorded on 100 randomly selected sperm nuclei per semen sample evaluated. There was no difference relative to week 0 in the least squares mean or SD of HA0, HA1, HA2, HA3, HA4, or HA5 over the 8 weeks (P > 0.05). However stallions were consistently different for all HAs (P < 0.05). The overall mean HA0–HA5 � SEM were 1.973 � 0.38, 0.087 � 0.002, 0.721 � 0.021, 0.051 � 0.004, 0.208 � 0.014, and 0.031 � 0.002, respectively. Even though libido increased during the experiment, confirming the effect of light on the stallions, no affect on sperm nuclear shape or its variation was detected using FHA. Based on work in other species involving numbers of sperm inseminated, if large numbers of sperm from these stallions were inseminated in mares, we would predict no change in fertility due to season. Further research is needed to confirm this prediction.

21 Effect of egg yolk extracted low-density lipoprotein on cryopreserved Nguni bull semen

2019 ◽  
Vol 31 (1) ◽  
pp. 136
Author(s):  
M. M. Tshabalala ◽  
K. A. Nephawe ◽  
M. L. Mphaphathi ◽  
C. M. Pilane ◽  
T. L. Nedambale
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Semen Parameters ◽  
Dna Integrity ◽  
Low Density ◽  
Bull Semen ◽  
Frozen Semen ◽  
Live Sperm

Egg yolk has been reported to have a beneficial effect on sperm quality following cryopreservation, and this led to its widespread use in semen extenders. However, egg yolk contains substances that inhibit respiration of sperm cells and diminish their motility rate. Moreover, it also contains low-density lipoproteins (LDL) that have a protective effect on sperm during the cryopreservation process. The objective of this study was improve cryopreservation of Nguni bull semen using egg yolk low-density protein. A total of 25 ejaculates were collected from 5 Nguni bulls aged 4 to 5 years using an electroejaculator during the natural breeding season. Collected raw semen samples were transported to the laboratory and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity before dilution. Semen was randomly diluted with a sodium citrate extender supplemented with 20% egg yolk (control) and with 6, 8, 10, and 12% LDL concentrations. The diluted semen sample groups were equilibrated for 4h at 5°C. Following equilibration, semen was loaded into 0.25-mL straws and frozen in a controlled-rate programmable freezer. The groups of semen straws were then plunged into LN and transferred into LN tanks (−196°C) for storage. The frozen semen straws per treatment group were thawed at 37°C and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity. Data were analysed with ANOVA using Stata V12 statistical software (StataCorp., College Station, TX), and treatment means were separated using Fisher’s protected t-test at the significant level of P&lt;0.05. Sperm motility and membrane integrity were significantly higher (P&lt;0.05) in frozen-thawed semen diluted with 8% LDL compared with the other concentrations. However, 6 and 8% LDL resulted in a significantly higher (P&lt;0.05) live sperm, DNA, and acrosome integrity. Frozen-thawed semen diluted with 10 and 12% LDL resulted in the lowest percentages of sperm motility, live sperm, plasma membrane, acrosome, and DNA integrity following cryopreservation. In conclusion, extender containing 8% LDL resulted in improved Nguni bull semen parameters such as sperm motility, viability, plasma membrane, acrosome, and DNA integrity following cryopreservation. Further studies are required to determine the fertilizing capacity of semen diluted and frozen with LDL in vitro and in vivo.

scholarly journals Sexual selection on protamine and transition nuclear protein expression in mouse species

2014 ◽  
Vol 281 (1783) ◽  
pp. 20133359 ◽  
Author(s):  
Lena Lüke ◽  
Polly Campbell ◽  
María Varea Sánchez ◽  
Michael W. Nachman ◽  
Eduardo R. S. Roldan
Keyword(s):  
Sexual Selection ◽  
Sperm Competition ◽  
Nuclear Protein ◽  
Sperm Head ◽  
Nuclear Proteins ◽  
Sperm Chromatin ◽  
Head Shape ◽  
Mouse Species ◽  
The Relationship ◽  
Protamine 2

Post-copulatory sexual selection in the form of sperm competition is known to influence the evolution of male reproductive proteins in mammals. The relationship between sperm competition and regulatory evolution, however, remains to be explored. Protamines and transition nuclear proteins are involved in the condensation of sperm chromatin and are expected to affect the shape of the sperm head. A hydrodynamically efficient head allows for fast swimming velocity and, therefore, more competitive sperm. Previous comparative studies in rodents have documented a significant association between the level of sperm competition (as measured by relative testes mass) and DNA sequence evolution in both the coding and promoter sequences of protamine 2. Here, we investigate the influence of sexual selection on protamine and transition nuclear protein mRNA expression in the testes of eight mouse species that differ widely in levels of sperm competition. We also examined the relationship between relative gene expression levels and sperm head shape, assessed using geometric morphometrics. We found that species with higher levels of sperm competition express less protamine 2 in relation to protamine 1 and transition nuclear proteins. Moreover, there was a significant association between relative protamine 2 expression and sperm head shape. Reduction in the relative abundance of protamine 2 may increase the competitive ability of sperm in mice, possibly by affecting sperm head shape. Changes in gene regulatory sequences thus seem to be the basis of the evolutionary response to sexual selection in these proteins.

Colloid single-layer centrifugation improves post-thaw donkey (Equus asinus) sperm quality and is related to ejaculate freezability

2015 ◽  
Vol 27 (2) ◽  
pp. 332 ◽  
Author(s):  
I. Ortiz ◽  
J. Dorado ◽  
D. Acha ◽  
M. J. Gálvez ◽  
M. Urbano ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Single Layer ◽  
Quality Parameters ◽  
Sperm Parameters ◽  
Computer Assisted ◽  
Sperm Analysis ◽  
Equus Asinus ◽  
The Relationship ◽  
Fresh Semen

The aim of this study was to determine whether colloid single-layer centrifugation (SLC) improves post-thaw donkey sperm quality and if this potential enhancement is related to ejaculate freezability. Semen from Andalusian donkeys was frozen following a standard protocol. SLC was performed on frozen–thawed semen and post-thaw sperm parameters were compared with uncentrifuged samples. Sperm quality was estimated by integrating in a single value sperm motility (assessed by computer-assisted sperm analysis), morphology and viability (evaluated under brightfield or fluorescence microscopy). Sperm freezability was calculated as the relationship between sperm quality obtained before freezing and after thawing. Ejaculates were classified into low, medium and high freezability groups using the 25th and 75th percentiles as thresholds. All sperm parameters were significantly (P < 0.01) higher in SLC-selected samples in comparison to uncentrifuged frozen–thawed semen and several kinematic parameters were even higher than those obtained in fresh semen. The increment of sperm parameters after SLC selection was correlated with ejaculate freezability, obtaining the highest values after SLC in semen samples with low freezability. We concluded that, based on the sperm-quality parameters evaluated, SLC can be a suitable procedure to improve post-thaw sperm quality of cryopreserved donkey semen, in particular for those ejaculates with low freezability.

Determining the relationship between bull sperm kinematic subpopulations and fluorescence groups using an integrated sperm quality analysis technique

2018 ◽  
Vol 30 (6) ◽  
pp. 919 ◽  
Author(s):  
J. L. Yániz ◽  
I. Palacín ◽  
K. S. Caycho ◽  
C. Soler ◽  
M. A. Silvestre ◽  
...  
Keyword(s):  
Staining Method ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Analysis ◽  
Kinematic Data ◽  
Analysis Technique ◽  
Analysis Of Results ◽  
Holstein Bulls ◽  
The Relationship

The aim of the present study was to determine whether there is an association between the kinematic sperm subpopulations and fluorescent groups in bulls using a new fluorescent staining method that allows classification of spermatozoa into groups depending on their acrosomal and membrane integrity, as well as functional status, without inhibiting sperm motility. Cryopreserved semen samples from 10 Holstein bulls were used in the study. A multiparametric analysis of results obtained by the ISAS 3Fun kit (Proiser) was performed. The different fluorescent groups were detected and their motility characteristics evaluated using ISAS software. Clustering procedures using the kinematic data resulted in the classification of spermatozoa into three kinematic sperm subpopulations. The distribution of kinematic sperm subpopulations was different between the fluorescent sperm groups (P < 0.001), although the correlation between them was low (r = 0.113; P < 0.01).

Selenium in blood, semen, seminal plasma and spermatozoa of stallions and its relationship to sperm quality

2010 ◽  
Vol 22 (5) ◽  
pp. 886 ◽  
Author(s):  
H. Bertelsmann ◽  
S. Keppler ◽  
M. Höltershinken ◽  
H. Bollwein ◽  
D. Behne ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Selenium Concentration ◽  
Selenium Level ◽  
Sperm Chromatin ◽  
Glutathione Peroxidase Activity ◽  
Progressive Motility ◽  
Three Samples ◽  
The Relationship

The essential trace element selenium is indispensable for male fertility in mammals. Until now, little data existed regarding the relationship between selenium and sperm quality in the stallion. Selenium, or selenium-dependent glutathione peroxidase activity, was determined in red blood cells, semen, seminal plasma and spermatozoa, and the percentages of spermatozoa with progressive motility (PMS), intact membranes (PMI), altered (positive) acrosomal status (PAS) and detectable DNA damage, determined by the sperm chromatin structure assay, were evaluated in 41 healthy stallions (three samples each). The pregnancy rate per oestrus cycle (PRC) served as an estimation of fertility. An adverse effect on stallion fertility caused by low dietary selenium intake was excluded, as all stallions had sufficient selenium levels in their blood. Interestingly, no significant correlations (P > 0.05) between the selenium level in blood and the selenium level in seminal plasma or spermatozoa were found, suggesting that the selenium level in blood is no indicator of an adequate selenium supply for spermatogenesis. The selenium level in spermatozoa (nmol billion–1) was correlated with PMI, PMS and PAS (r = 0.40, r = 0.31 and r = –0.42, respectively; P ≤ 0.05), and the selenium concentration in spermatozoa (nmol g–1) was correlated with PRC (r = 0.40, P < 0.03). The results of the present study show that the determination of an adequate selenium status for the male equine reproduction requires the analysis of selenium in spermatozoa. Furthermore, selenium is associated with improved sperm quality and fertility in the stallion.

First report on in vivo fertility trial of frozen thawed boar semen in India#

2016 ◽  
Author(s):  
S. K. Baishya ◽  
R. K. Biswas ◽  
G. Kadirvel ◽  
B. C. Deka ◽  
Suresh Kumar ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Sperm Quality ◽  
Quality Parameters ◽  
Frozen Semen ◽  
Boar Semen ◽  
The Mean ◽  
Live Sperm ◽  
Farrowing Rate ◽  
Size At Birth

The present study was conducted to evaluate the in vivo fertility of frozen thawed boar semen. Twenty ejaculates collected from six mature boars were frozen in a programmable freezer. After freezing the semen was evaluated for different sperm quality parameters. Twenty five sows were inseminated artificially utilizing frozen thawed semen. The percentage of sperm motility, live sperm, live intact acrosome, plasma membrane intact sperm, HOST-reacted sperm, live sperm with high mitochondrial membrane potential, lipid peroxidised sperm and DNA-damaged sperm of frozen semen utilised for AI were 56.25 ± 0.96, 63.75 ± 1.47, 59.88 ± 1.09, 41.08 ± 1.01, 40.31 ± 1.02, 86.23 ± 1.29, 9.28 ± 0.83 and 4.20 ± 0.29 respectively. The farrowing rate was 44.00 per cent and the mean litter size at birth was 5.91 ± 0.69. It could be concluded that the freezing and insemination protocol of boar semen used in the present study resulted in moderate fertility of frozen boar semen and it would help in further improvement and utilization of frozen boar semen for AI in India.

scholarly journals Cryopreservation and quality assessment of boar semen collected from bulk samples

2019 ◽  
Vol 64 (No. 5) ◽  
pp. 209-216 ◽  
Author(s):  
R Ratchamak ◽  
T Vongpralub ◽  
W Boonkum ◽  
V Chankitisakul
Keyword(s):  
Sperm Motility ◽  
Semen Quality ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Bulk Sample ◽  
Energy Status ◽  
Semen Volume ◽  
Boar Semen ◽  
Live Sperm ◽  
Fresh Semen

The purpose of this study was to examine sperm quality after cryopreservation of ejaculates collected as a bulk sample, which is routinely part of semen collection, and to compare this quality with the sperm-rich fraction in boars. Ejaculates were collected as sperm-rich fractions (SRF) and bulk samples (BE) using a gloved-hand technique. Fresh semen quality in terms of semen volume, sperm concentration, total sperm motility and pH were conventionally evaluated. Then, semen was cryopreserved using the liquid nitrogen vapour method. The post-thaw sperm quality was evaluated by assessing sperm motility, live sperm with normal apical ridge and high mitochondrial energy status, lipid peroxidation was evaluated using CASA and fluorescent multiple staining and MDA levels were determined using a spectrophotometer, respectively. In terms of fresh semen quality, sperm motility in fresh semen did not differ significantly between the two groups. The treatment with the greater mean volume (BE; P &lt; 0.05) had a lower mean sperm concentration (P &lt; 0.05); meanwhile, the mean ejaculate pH collected as BE was more basic compared with SRF (P &lt; 0.05). However, there were no significant post-thaw quality changes between sperm-rich fractions and bulk samples of semen. In conclusion, ejaculates can be collected as bulk samples without the need to classify fractions for boar semen cryopreservation.

scholarly journals Seminal Attributes, Freezability and their Interrelationships in Zebu Cattle and Buffalo Bulls from Central Gujarat

2018 ◽  
Vol 14 (2) ◽  
Author(s):  
P. K. Pathak ◽  
A. J. Dhami ◽  
D. V. Chaudhari
Keyword(s):  
Semen Quality ◽  
Conventional Technique ◽  
Zebu Cattle ◽  
Motile Sperm ◽  
Morphological Attributes ◽  
Frozen Semen ◽  
The Mean ◽  
Live Sperm ◽  
Sperm Output ◽  
Fresh Semen

A study was carried out on nine healthy mature breeding bulls (3 each of Gir, Surti and Murrah breed) to evaluate their fresh and frozen semen quality and their interrelationships. The ejaculates immediately after collection were evaluated for routine physico- morphological attributes, including HOS test. The ejaculates (n=72) having >75% initial motility were diluted @ 80 million sperm/ml using TFYG extender and the French mini straws filled were frozen in liquid nitrogen vapour using a programmable biofreezer. Thawing of straws was done at 37°C for 30 sec and assessed for freezability by conventional technique. All the cattle and buffalo bulls donated consistently normal thick creamy yellow and thick milky white semen, respectively. In Gir, Suti and Murrah bulls (n=24 ejaculate each) the seminal attributes such as ejaculate volume (6.69±0.17, 3.12±0.10 and 3.96±0.16 ml, p less than 0.01); initial motility (80.21±0.88, 84.58±0.60 and 84.38±0.76 %, p less than 0.01); total sperm output/ejaculate (9013.85±265.32, 3935.49±259.63 and 5366.48±332.99 million, p less than 0.01) and live sperm (84.71±0.83, 86.17±0.78 and 86.79±0.79 %, p less than 0.05) differed significantly. The mean percentages of post-thaw motile sperm (53.29±1.56, 58.33±1.43 and 59.58±1.20, p less than 0.01); live sperm (59.00±1.95, 67.00±1.59 and 68.42±1.66 %, p less than 0.01); and HOS reactive sperm (48.25±0.78, 44.21±1.29 and 51.54±1.29 %, p less than 0.01) in Gir, Surti and Murrah bulls semen also differed significantly. The variation among the bulls was significant for buffalo breeds in most of their fresh seminal attributes, except HOST, and for post-thaw motility, but not among Gir bulls. The important seminal attributes like motility, live sperm and HOS reactive sperm of fresh and frozen-thawed semen were significantly and positively interrelated in all three breeds of bulls (r = 0.40 to 0.81, p less than 0.05 to 0.01), suggesting that motility and HOST of fresh semen were good predictors of freezability of bovine semen.

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